Quantum dots in modular nucleic acid scaffolds operable as nanoscale energy harvesting and focusing arrays

ABSTRACT

The invention relates to a nanoscale antenna including a nucleic acid scaffold having a structure selected from the group consisting of a Holliday junction, a star, and a dendrimer; and a plurality of fluorophores attached to the scaffold and configured as a FRET cascade comprising at least three different types of fluorophores including at least one quantum dot, arranged with (a) a plurality of initial donor fluorophores fixed in exterior positions on the structure, (b) a terminal acceptor fluorophore fixed in a central position on the structure, and (c) a plurality of intermediate fluorophores fixed in positions on the scaffold between the initial acceptor fluorophores and the terminal acceptor fluorophores.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit as a division of U.S. patentapplication Ser. No. 14/685,836 filed Apr. 14, 2015 which in turn claimsthe benefit of U.S. Provisional Application No. 61/979,727 filed on Apr.15, 2014, the entirety of each of which is incorporated herein byreference.

BACKGROUND

A need exists for techniques to focus light excitonic energy and tostudy Förster resonance energy transfer (FRET) phenomena.

BRIEF SUMMARY

In a first embodiment, nanoscale antenna includes a nucleic acidscaffold having a structure selected from the group consisting of aHolliday junction, a star, and a dendrimer; and a plurality offluorophores attached to the scaffold including at least one quantum dotand configured as a FRET cascade comprising at least three differenttypes of fluorophores, arranged with (a) a plurality of initial donorfluorophores fixed in exterior positions on the structure, (b) aterminal acceptor fluorophore fixed in a central position on thestructure, and (c) a plurality of intermediate fluorophores fixed inpositions on the scaffold between the initial acceptor fluorophores andthe terminal acceptor fluorophores.

In another embodiment, the scaffold of the nanoscale antenna of thefirst embodiment has a dendrimer structure.

In a further embodiment, a method includes exciting the antenna of thefirst embodiment with a light source.

In an additional embodiment, one or more portions of said scaffoldincorporating intermediate fluorophores includes a toehold sequence, aredetachable from the remaining portion of said scaffold upon contact witha sequence complementary to the toehold sequence.

Aspects of the invention, including details on techniques and additionalexemplary DNA sequences, are described in Buckhout-White et al.,“Assembling programmable FRET-based photonic networks using designer DNAscaffolds,” Nature Communications 5, Article number:5615,doi:10.1038/ncomms6615 and the associated supplementary material, theentirety of which is incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1F depict exemplary DNA structures and fluorophorephotophysical properties. FIG. 1A shows 2-dye single FRET step systemconsisting of Cy3 donors paired to a Cy5 acceptor. The ratio ofCy3_(n)/Cy5 was incrementally increased using linear (n=1), bifurcated(n=2), Holliday junction (n=4), and star (n=8) structures.Donor-acceptor spacings are also varied for each as an increments of theFörster distance (0.75, 0.87, 1.0, 1.25, 1.5×R₀˜54 Å). The 1.25×R₀structures show dye locations relative to the DNA. FIG. 1B shows 4-dye,three FRET step system with sequential donor-acceptor arrangements ofCy3, Cy3.5, Cy5, Cy5.5 in photonic wire configurations. The number of[Cy3→Cy3.5→Cy5]_(n) wires leading into each terminal Cy5.5 dye increasesfrom 1 to 8 using linear, bifurcated, Holliday junction and 8-arm starconstructs. The blue arrows show the directionality of the FRETcascade(s) in each structure. Donor-acceptor spacing varied here at 0.5,1.0, 1.5×R₀. The 1.5×R₀ schematic shows the dye positions. FIG. 1C showsdendrimer-based FRET systems utilizing Cy3, Cy3.5, Cy5, Cy5.5 dyes inconfigurations were each dye preceding the central-terminal Cy5.5 has2-, 3-, or 4-donors. Donor-acceptor spacings for the dendrimers werefixed at 0.5×R₀ and the 2:1 structures show dye locations. FIG. 1D showsdendrimer-based 5-dye FRET system utilizing AF488, Cy3, Cy3.5, Cy5,Cy5.5 dyes in configurations where each dye preceding thecentral-terminal Cy5.5 has 2-donors. An alternate version was assembledwith AF647 replacing Cy5. Blue arrows schematically highlight thegeneral donor-to-acceptor architecture. FIG. 1E shows normalizedabsorption/emission spectra for each of the dyes used. FIG. 1F plots theintegrand of the J integral as a function of wavelength for pertinentdonor-acceptor combinations.

FIGS. 2A-2D illustrate exemplary single FRET systems using 2 dyes. FIG.2A shows representative spectra excited at 515 nm showing the effect ofaltering Cy3-Cy5 donor acceptor spacing as a function of R₀˜54 Å. Thedirect Cy3 only emission in each was used to normalize the data. FIG. 2Bshows spectra from linear, bifurcated, Holliday junction and 8-arm starCy3-Cy5 constructs where donor-acceptor spacing was maintained at˜0.75×R₀. Data were normalized to the Cy5 alone emission. FIG. 2C is aplot of the Cy5 terminal enhancement factor (TEF) as a function of thenumber of Cy3 donors/Cy5 acceptor for each of the donor-acceptorspacings as compared to the initial 1.5×R₀ Cy3₁→Cy5 system. Trend linesare added to aid the eye. FIG. 2D shows single pair FRET (spFRET)histograms for all 0.75×R₀ constructs, see SI for methodology. Thenumber of FRET events for each curve has been normalized to 1. Thegreen, blue and red, curves are shifted for presentation. Note thegrowth of the 0 FRET efficiency curve (representing a subpopulation thatis not undergoing FRET) in the star structure and the shift to higherefficiency from linear to the star structure.

FIGS. 3A-3H illustrate exemplary 4-dye photonic wire and dendrimersystems. FIG. 3A illustrates representative data showing the spectralevolution of the 0.5×R₀ linear Cy3→Cy5.5 system as consecutive acceptordyes are added to the initial Cy3 donor. FIG. 3B shows comparativespectral data for the 0.5, 1.0 and 1.5×R₀ linear systems. Data werenormalized to the direct Cy5.5 emission at the same excitation. Inset,composite and deconvolved individual component spectra for the 0.5×R₀linear system. FIGS. 3C-3E show spectra showing the FRET evolution ofthe (C) 1.5×R₀, (D) 1.0×R₀, and (E) 0.5×R₀ systems as the number of armsfor each was increased from one to four. The inset in C-D pots theincrease in Cy5.5 peak emission as a function of the number of arms allon the same scale. FIG. 3F shows comparative spectral data for the fullyassembled 2:1, 3:1, and 4:1 0.5×R₀ dendrimer structures. Stoichiometryfor 2:1 structure=Cy3₈→Cy3.5₄→Cy5₂→Cy5.5₁; 3:1structure=Cy3₂₇→Cy3.5₃→Cy5₃→Cy5.5₁; 4:1structure=Cy3₆₄→Cy3.5₁₆→Cy5₄→Cy5.5₁. Note all data in C—H werenormalized to the direct Cy5.5 emission. Spectral data following theevolution of the AF488₁₆→Cy3₈→Cy3.5₄→Cy5₂→Cy5.5₁ (FIG. 3G) andAF488₁₆→Cy3₈→Cy3.5₄→AFF647₂→Cy5.5₁ (FIG. 3H) 2:1 dendrimer systems.Constructs with Cy3 initial dye were excited at 515 nm while those withAF488 were excited at 465 nm.

FIGS. 4A-4D show energy transfer in the photonic wire and dendrimersystems. FIG. 4A is a plot of the Cy5.5 terminal enhancement factor(TEF) for the [Cy3→Cy3.5→Cy5]_(n)→Cy5.5 photonic wire and the 2:1, 3:1,and 4:1 0.5×R₀ dendrimer structures as compared to the initial 1.5×R₀linear system. Note the break in vertical scale. FIG. 4B shows acomparison of the normalized emission profiles for the 0.5×R₀ 2:1dendrimer and 8-arm photonic wire star structures. Dye ratioscorresponding to each position in each structure are indicated with redor blue. Note the significant deconvolved Cy5.5 sensitization for the2:1 dendrimer-inset. FIG. 4C is a comparative plot of the sensitizedcomponents at each step for the [Cy3→Cy3.5→Cy5]_(n)Cy5.5 photonic wiresystem. Dye emissions are scaled and normalized to the highestcomponent, the Cy3.5 sensitized emission in the 8-arm star structure.FIG. 4D is a comparative plot of the sensitized components at each stepfor the 0.5×R₀ 2:1, 3:1, and 4:1 dendrimer system. Dye emissions arescaled and normalized to the highest component, the Cy3.5 sensitizedemission in the 4:1 structure.

FIGS. 5A-5D show energy transfer analysis, namely a comparison ofexperimental data (circles) with spectra predicted by “ideal” simulation(lines) for 1-, 2-, 4-, and 8-arm multi-dye structures with dye spacingsof (A) 1.5×R₀, (B) 1.0×R₀, and (C) 0.5×R₀, and (D) for dendrimers withdye spacing of 0.5×R₀ and branching ratios of 2:1, 3:1 and 4:1. Ingeneral, agreement between experiment and “ideal” simulation worsens asdye spacing gets smaller.

FIGS. 6A-6D show calculated ideal yield and efficiency. FIG. 6A showsthe yield of target structures as assessed by gel electrophoresis orFPLC for multi-dye photonic wire structures and dendrimers with 0.5×R₀dye spacing versus the number of arms or branching ratio, as comparedwith the corresponding yields derived from fitting the PL spectra whenthe structures are functionalized with either three or four dyes. FIG.6B shows actual and ideal anywhere-to-end efficiency computed for the4-dye linear structures as a function of the number of arms with the dyespacing as a parameter. FIG. 6C shows actual and ideal end-to-endefficiency computed for dendrimers as a function of branching ratio.Highlighting the importance of parallel FRET pathways, three idealcurves are shown, one assuming only nearest neighbor FRET (inset, left),one including only intra-arm FRET (inset, right), and one including allFRET processes (Ideal). FIG. 6D shows actual and ideal antenna gains ascomputed for the four-dye linear structures and dendrimers with 0.5×R₀dye spacing. Note, the 0.5×R₀ linear photonic wire structure correspondsto the 1 arm dendrimer.

FIGS. 7A-7D schematically illustrate an exemplary dendrimer-based DNAsensor. FIG. 7A is a schematic of a [[Cy3₂-Cy3.5]₂-Cy5]₂-Cy5.5 2:1dendrimer where a toehold has been added onto the Cy3.5 containingoligonucleotide. FIG. 7B illustrates how, when exciting the initial Cy3donor, this structure might efficiently direct exciton energy to theterminal Cy5.5 acceptor through multiple overlapping FRET pathways. Onlythe direct spectrally- and spatially-favored FRET pathways are shownhere. FIG. 7C shows how the addition of the complimentary strand to thestructure in FIG. 7A (in a room temperature isothermal transition)removes the Cy3.5 labeled strands from the structure. FIG. 7D shows thatthis results in a significant decrease in the amount of excitonic energybeing delivered to the Cy5.5 terminal acceptor along with the magnitudeof its signal—even when excited at the Cy5 donor preceding it.

FIGS. 8A and 8B show representative photophysical characterization ofsome of the structures in FIGS. 7A-7D. FIG. 8A shows the antenna effect(AE) derived by comparing the emission of the terminal Cy5.5 when thesystem is excited at the initial Cy3 donor (515 nm) as compared toexciting the Cy5 donor (635 nm) preceding the Cy5.5. This is describedas AE=Cy5.5 PL(exc. 515)/=Cy5.5 PL (exc. 635). This signal enhancementreflects how much more the Cy5.5 is emitting when the system is excitedat the outer Cy3 dyes (n=8) than at the Cy5 dyes (n=2) preceding theCy5.5 acceptor. In this case it is around 2.5 times more. Addition ofthe complement, which removes the Cy3.5 intermediary, decreases thissignal enhancement significantly. This directly shows how much moreCy5.5 signal and change in signal is available for a potential sensingevent using the DNA antenna to sensitize the terminal acceptor. FIG. 8Bshows the initial Cy3 and terminal Cy5.5 deconvoluted signal in the fullstructure when excited at 515 nm (Cy3) and after removing the Cy3.5intermediary. Note the relative changes in emission and emission ratioswhich would be directly available to enhance the available signal andchange in signal for a biosensing event.

DETAILED DESCRIPTION

Definitions

Before describing the present invention in detail, it is to beunderstood that the terminology used in the specification is for thepurpose of describing particular embodiments, and is not necessarilyintended to be limiting. Although many methods, structures and materialssimilar, modified, or equivalent to those described herein can be usedin the practice of the present invention without undue experimentation,the preferred methods, structures and materials are described herein. Indescribing and claiming the present invention, the following terminologywill be used in accordance with the definitions set out below.

As used in this specification and the appended claims, the singularforms “a”, “an,” and “the” do not preclude plural referents, unless thecontent clearly dictates otherwise.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items.

As used herein, the term “about” when used in conjunction with a statednumerical value or range denotes somewhat more or somewhat less than thestated value or range, to within a range of ±10% of that stated.

Overview

Described herein is a technique for the modular assembly of energytransfer moieties (namely fluorophores such as fluorescent dyes) into ananoscale antenna such that energy (light) is absorbed at some discretepoint(s) and funneled in a specific direction, such as inward and/oroutward from a central point via Förster resonance energy transfer(FRET).

The fluorophores are fixed in positions on a nucleic acid scaffold, forexample a modular scaffold, preferably a DNA scaffold comprisingoligonucleotide elements that assemble on the basis of nucleic acidcomplementarity.

Some embodiments have a single terminal acceptor flurophore, howevertrials with more than one terminal acceptor flurophore were found toincrease the overall energy transfer efficiency through the system.Multiple terminal acceptors increase the probability that the excitonwill be transferred to the terminal dye(s), especially if the energy isbeing funneled through multiple pathways.

More than simple linear arrangements, this technique utilizesmulti-fluorescent molecular configurations in such a way that energy istransferred though multiple, discrete steps as desired. Furthermore, thearrangement of these molecules and the number of steps involved can bequickly modified through inclusion or exclusion of specific DNA strandsallowing for many different permutations to be created within one sampleset. Fundamental to the activity of these structures, termed nanoscaleantennas, is the ability to harvest light energy and then concentrate itor focus/direct it in a particular direction. It is expected that suchnanoscale antennas could be incorporated into active nanodevices toprovide power or other functionalities.

Excitation of a nanoscale antenna may occur using a instrument, forexample with a laser, or otherwise. For example, it may operatefollowing chemical excitation such as that of bioluminescent resonanceenergy transfer (BRET) where an enzyme such as luciferase chemicallygenerates the light from oxidizing substrate. Alternately, the antennamay operate on the basis chemically generated light.

Suitable fluorophores can by site-specifically fixed to the nucleicacid, preferably by covalent attachment. Possible modes of attachmentfor fluorophores include using (a) phosphoramidite chemistry, with dyesinserted directly into the scaffold by end-labeling or by placing thembetween two bases and opposite, for example, an unpaired A base; (b)succinimidyl ester chemistry, e.g., by attachment to an amine-modifiedlinker placed either 5′, 3′ or inserted internally; (c) maleimide thiolchemistry; (d) carboxyl-amine amide bond formation by carbodiimidechemistry; (e) azide-alkyne cycloaddition; and (f) electrostaticbinding, as well as combinations thereof. Other suitable attachmentmechanisms can be contemplated by one of skill in the art.

The demonstrated structures vary the distance between the fluorophoresaccording to their Förster resonance energy transfer (FRET) distance(where R₀ denotes the Förster distance which is the distance where 50%FRET efficiency occurs). The distances between fluorophores can becontrolled as desired when designing the scaffold.

Various types of structures were designed, assembled, tested andanalyzed. The first of these structure types, the 2-dye system, aimed toisolate the effect of the multiple donors separate from the multiple-dyecascading FRET. For this system, a well-characterized Cy3-Cy5 donoracceptor pair was used. In an effort to understand the potential trendsseen by increasing the number of donor with reference to a singleacceptor, the system assembled dyes according to a D_(n)-A formula,where n=1, 2, 4 or 8 and where each D individually represents afluorophore donor and where A represents an acceptor, with all the Dfluorophores apart from D1 also operable as intermediatedonor/acceptors. This led to the creation of a unidirectional linearstructure, a bifurcated linear structure, an intersecting structurebased on a Holliday junction and an 8-armed star structure. Thesestructures represent a truly modular system in that all versions withina given distance were made using the same set of 10 strands of DNA.

The linear and bifurcated structures are made from a simple doublestranded piece of DNA. The Cy5 dye is internally labeled at the centerof one of the oligos and the 3′, for the linear structures—3′ and 5′ends for the bifurcated are labeled with a Cy3. For the Hollidaystructure, an internally labeled Cy5 is placed on the center of one offour single-stranded oligos. Two of the three remaining oligos arelabeled at both the 3′ and 5′ ends with Cy3 and a fourth oligo isunlabeled. The star is constructed similarly to the Holliday structurewith a central, internally labeled Cy5, 4 double Cy3-labeled strands and3-unlabeled strands. A variety of spacing were investigated including0.75-, 0.87-, 1.0-, 1.25- and 1.5×R₀. This was accomplished by changingthe length of the DNA arms.

Exemplary oligonucleotide elements used to create these structures atthe 1.5×R₀ spacing are as follows, where an asterisk “*” represents thelocation of a nucleotide modification to incorporate a dye:

T1 (SEQ ID No: 1; 5′ Cy3, 3′ Cy3)*GGAGAGATGGTTCAGCCGCAATCCTCGCCTGCACTCTACCTGACTTC C*T2 (SEQ ID No: 3; Internal Cy5)GGAAGTCAGGTAGAGTGCAGGCGA*GAGCACGAGTCTTGCTGCTTAGC T3 (SEQ ID No 3; 5′Cy3, 3′ Cy3) *GCTAAGCAGCAAGACTCGTGCTCACCGAATGCCACCACGCTCCGTCG C*T4 (SEQ ID No: 4) GCGACGGAGCGTGGTGGCATTCGGCGTCCAGCTCTGATCCAATACTCCT5 (SEQ ID No: 5; 5′ Cy3, 3′ Cy3)*GGAGTATTGGATCAGAGCTGGACGACAATGACGTAGGTCCTAACCTC C* T6 (SEQ ID No 6):GGAGGTTAGGACCTACGTCATTGTACTATGGCACACATCCCTAGTTCC T7 (SEQ ID No: 7; 5′Cy3, 3′ Cy3) *GGAACTAGGGATGTGTGCCATAGTGGTCAACGCATACACCTTCTATC C*T8 (SEQ ID No: 8) GGATAGAAGGTGTATGCGTTGACCGGATTGCGGCTGAACCATCTCTCCT9 (SEQ ID No: 9) GCGACGGAGCGTGGTGGCATTCGGGGATTGCGGCTGAACCATCTCTCCT10 (SEQ ID No: 10; Internal Cy5)GGAGGTTAGGACCTACGTCATTG*CGTCCAGCTCTGATCCAATACTCC

The “eight way” star structure eight sequences T1-T8. The “four-way”Holliday star used T1, T2, T3, and T9. The linear structures used T5 andT10.

Moving to a more complex 4-dye system expands to include the foursequentially arrayed dye cascade of Cy3, Cy3.5, Cy5 and Cy5.5. Thissystem is designed to assemble the dyes similar to the 2-dye systemabove in a D1_(n)-D2_(n)-D3_(n)-A, where n equals 1, 2, 4, or 8 andwhere each D individually represents a fluorophore donor and where Arepresents an acceptor, with all the D fluorophores apart from D1 alsooperable as intermediate donor/acceptors. These structures are measuredat three different Förster distances (0.5-, 1.0- and 1.5×R₀). The 1.0-and 1.5×R₀ spacing structures are designed much the way of the 2-dyesystems. To account for the additional space needed for the cascadingFRET dyes, one side of the double stranded arm is extended. This occurson the linear and bifurcated structures and on each arm of the Hollidayand star structures. The extended arms then act as a template for thesmaller dye-labeled oligos to be assembled. Given the close distancesneeded in the 0.5×R₀ spacing, the assembly proceeds differently. For thelinear structure, 4 strands concatenate together to form the structureand each of these strands contains one dye. The bifurcated structurealso uses concatenated strands but requires the Cy5 to be double-labeledand one of the oligos to be double-labeled with Cy3 on the 3′ end andCy5.5 on the 5′ end. All other oligos contain one dye at either end orinternally-labeled. The Holliday and star structures contain an internalCy5.5 at the center junction. Two of the remaining central oligos in theHolliday and four for the star contain double internally labeled Cy5dyes. The Cy3 and Cy3.5 are assembled similar to the 1-way structures ina concatenated manner at the end of the central oligos.

In the 4-dye system, the first three dyes engage in a 1:1correspondence. Another set of DNA structures described here aimed tocreate a system where each acceptor has multiple donors according to thefollowing: D1_(n) ³-D2_(n) ²-D3_(n)-A, where n equals 2, 3, or 4 andwhere each D individually represents a fluorophore donor and where Arepresents an acceptor, with all the D fluorophores apart from D1 alsooperable as intermediate donor/acceptors. In order to facilitate thistype of structure an exponential branching motif is used. The dendrimersystem in the center begins with a branch of n arms and then eachsubsequent branch contains n+1 arms. The structures are designed with 2long 58 base oligos, one of which is internally labeled at the centerwith Cy5.5 and the other has 2 internal Cy5 labels that occur at thebranching junction. 40 base oligos are assembled to the 58 base centerand are double, internally-labeled with Cy3.5. Finally the ends are 18base oligos with 3′ and 5′ labeled Cy3.

Spectral Overlap

The particular cyanine and Alexa Fluor dyes were chosen for study inpart because they are commercially available site-specifically attached(internal or terminal) to DNA. In addition, most have been usedpreviously in photonic wire work, thereby providing archival backgroundon their performance. The plot in FIG. 1E shows the dyeabsorption/emission spectra and highlights the potential of this systemto be excited at ˜515-550 nm for Cy3, and then to transfer excitonenergy in 1-step to Cy5 or in a 3-step FRET cascade to the terminalCy5.5. Alternatively, the AF488 can be excited at 460-490 nm andtransfer energy in a 4-step cascade to Cy5.5. Relevant photophysicalparameters are listed in Table I along with the calculated spectraloverlap integrals (J) and R₀ for each donor-acceptor pair. R₀ values foreach FRET pair varied between ˜40 to 70 Å while J varied almost an orderof magnitude from 9.4×10⁻¹³ to 1.2×10⁻¹² cm³M⁻¹. Values for potentialhomoFRET between like dyes are also listed. Plots of the integrand forthe J integral as a function of wavelength for pertinent donor-acceptorpairs are shown in FIG. 1F and reinforce the concept of transferringexciton energy sequentially over a ˜250 nm portion of the spectrum usingmultiple ET steps. The sequential nature of the system suggests thatlonger-range FRET, i.e. skipping over an intermediary dye, is not asfavorable.

Photophysical Performance

Cy3_(n)→Cy5 Single FRET Step Systems

This system was designed to evaluate the sensitization of a singleacceptor as function of the number of donating dyes and how this dependson the separation distance. For these experiments, the DNA scaffold wasused to array 1, 2, 4, or 8 Cy3 donors around a central Cy5 acceptor atseparation distances ranging from 0.75-1.5×R₀ (˜40-81 Å). Samples wereprepared and emission spectra collected with 515 nm excitation. FIG. 2Ashows representative, normalized spectra collected from the linear(Cy3₁→Cy5) system as the fluorophore distances were varied in comparisonto Cy3 alone. As expected, the Cy3 donor emission decreases and thecorresponding Cy5 acceptor sensitization increases as a function ofdecreasing separation. FIG. 2B presents normalized spectra from theclosest 0.75×R₀ assemblies as the ratio of Cy3 to Cy5 increases. DirectCy3 emission increases concomitant with its valence and Cy5sensitization also increases but appears to plateau.

Terminal enhancement factor (TEF), antenna effect (AE), and excitonend-to-end efficiency (E) are the metrics applied to characterizing theenergy harvesting/sensitizing capabilities in these systems. Theend-to-end energy transfer efficiency, E, of each construct wascalculated using the expression:E=[(Φ_(AD)−Φ_(A))/Q _(A)]/(Φ_(D) /Q _(D))where Φ_(AD) is the integrated PL area of the terminal acceptor in thepresence of donor, Φ_(A) is the integrated PL area of the terminalacceptor in the absence of donor, Φ_(D) is the integrated PL area of thedonor in the absence of acceptor, and Q_(A) and Q_(D) is the quantumyield (QY) of the terminal acceptor and donor, respectively. The value Eprovides a means to assess the terminal acceptor re-emission followingsensitization from an upstream fluorophore while also accounting for theQY of the initial donor and terminal acceptor. The values of Φ_(D) andΦ_(A) were determined by numerical integration of PL area fits frommolar equivalent samples containing only the donor or acceptor ofinterest, respectively. The value Φ_(AD) was determined by deconvolutionof the composite emission spectrum of a FRET construct into the PLintensity from each contributing fluorophore and numerical integrationin the manner described above. The end result was a series of PLintensity curves corresponding to each fluorophore within a FRETconstruct. The value Φ_(AD) is the integrated PL intensity of theterminal acceptor in the presence of the primary FRET donor.

In constructs possessing multiple donors, E accounts for energy that hasentered the system through the initial donor as well as from directexcitation of intermediate fluorophores serving as donors. For example,in the full Cy3-Cy3.5-Cy5-Cy5.5 construct the majority of energy isintroduced to the system via direct excitation of Cy3. However, someenergy is introduced through direct excitation of Cy3.5 and to a lesserextent Cy5. Therefore, the magnitude of E reflects the amount ofterminal acceptor sensitization arising from energy that has entered thesystem through any upstream fluorophore, of which the Cy3 emissionprovides the significant majority.

Similar analysis was employed to quantify the average FRET donorefficiency, E_(D), and acceptor re-emission efficiency, E_(A), for eachdonor-acceptor pair within a particular construct. Direct excitationspectra fits for each fluorophore (determined from molar equivalentcontrol samples) were subtracted from fit emission data sets to minimizecontributions to the composite PL intensity arising from directexcitation of fluorophores subsequent to the initial donor. Followingthe scaling/subtraction method outlined above, the resulting spectrawere deconvoluted to determine the contributions from the primary donoremission and the sensitized acceptor contribution in a particularconfiguration. These deconvoluted data were numerically integrated andused to calculate E_(D) and E_(A) for each donor-acceptor conjugate:E _(D)=1−F _(DA) /F _(D)andE _(A) =F _(AD) /F _(D)where F_(DA) is the integrated PL area of the donor is the presence ofacceptor, F_(D) is the integrated PL area of the donor is the absence ofacceptor, and F_(AD) is the integrated PL area of the acceptor in thepresence of donor. The value E_(D) and E_(A) represent the donor lossand acceptor sensitization, respectively. In constructs with multiplefluorophores, the efficiency for each donor-acceptor pair is analyzed asan independent step, regardless of whether the donor was directlyexcited and/or sensitized.

The antennae effect (AE) was measured for all systems and is definedhere as:AE _(Cy3→Cy5) =I _(Cy5,515 nm) /I _(Cy5,635 nm)orAE _(Cy3→Cy3.5→Cy5→Cy5.5) =I _(Cy5.5,515 mm) /I _(Cy5.5,685 nm)where I_(Cy5,515 nm), I_(Cy5,635 nm) and I_(Cy5.5,515 nm),I_(Cy5.5,685 nm) are the fluorescence intensities (deconvolved areaunder the curve) of the terminal Cy5 or Cy5.5 following excitation ofthe initial Cy3 donor at 515 nm and direct excitation at 650/700 nm,respectively.

The terminal enhancement factor (TEF) was introduced to allow for acomparison of the PL intensity of a terminal acceptor (Cy5 or Cy5.5)across all FRET constructs (linear, bifurcated, Holliday junction, 8-armstar and dendrimers). First, PL data were normalized using the intensityof the Cy3-DNA conjugate since molar equivalence was maintained acrossall data sets. This accounted for any instrument variation during datacollection. The intensity from the unidirectional 0.5×R₀ construct waschosen as unity and all other data scaled accordingly. A second scalingfactor was then introduced to account for presumptive number of activeCy3 dyes within a particular construct: 1 for linear, 2 for thebifurcated, 4 for the Holliday junction, 8 for the 8-arm star way linearand 2:1 dendrimer, 27 for the 3:1 dendrimer, and 64 for the 4:1dendrimer. The PL intensity of terminal acceptor, determined fromdeconvolution of composite spectra, was subjected to this normalizationand scaling procedure and tabulated. The terminal PL intensity with thelowest value (unidirectional, 1.5×R₀) was then set to unity and allother data scaled up by this value. The result is a series of normalizeddata points reporting the PL intensity of Cy5.5 in the fullCy3-Cy3.5-Cy5-Cy5.5 configuration while also accounting for the variouslinear and dendrimeric constructs. Enhancement of sensitization indifferent experimental constructs is then given in comparison to aninitial construct. TEF is applied across structures containing a singleor equivalent terminal acceptor dye regardless of donor number,configuration or donor-acceptor separation.

Loosely speaking, both TEF and AE are relative measures of performance,giving the degree of enhancements over either a “reference” system (TEF)or direct excitation of the terminal dye (AE), while E estimates theefficiency of exciton delivery. Each can be measured empirically,directly from experiment, or calculated as theoretical values. FIG. 2Cplots TEF as a function of the number of Cy3 donors per Cy5 acceptor foreach of the donor-acceptor spacings where the “reference” system is theCy5 sensitization of the 1.5×R₀ linear construct. The plot shows thatoutput can be magnified by >60 times by decreasing the inter-fluorophoreseparation to 0.75×R₀ and increasing the number of donors to four.Regarding the corresponding AE and E metrics given in Table 2, the0.75×R₀ Holliday junction manifests the best AE at nearly 300% while the0.75×R₀ bifurcated system achieves ≥50% E. Excepting the 0.75×R₀structures, AE generally increases with Cy3 donor number, while E valuesare relatively constant up to a ratio of 4 and then decrease in goingfrom 4 to 8.

[Cy3→Cy3.5→Cy5]_(n)→Cy5.5 Photonic Wires

This system is a generalization of the two-dye system of the previoussub-section into an antenna with more dyes, a larger collection area,and perhaps higher performance. FIG. 3A shows the spectral evolution ofthe 0.5×R₀ linear (photonic wire) system as consecutive acceptor dyesare added to an initial Cy3 donor. A clear loss of donor emission and aconcurrent sensitization of each acceptor dye are noted for each addedstep. FIG. 3B compares normalized spectra from the 1.5, 1.0, and 0.5×R₀linear Cy3→Cy5.5 systems confirming that closer dye spacingssignificantly improve FRET efficiency as expected. The data presented inFIGS. 3C-E correspond to assembling the initial [Cy3→Cy3.5→Cy5] portionas linear photonic wires with inter-dye spacings of 1.5, 1.0, and 0.5×R₀and compare the increase in wire valency from one to eight around theterminal Cy5.5, see FIG. 1B for structure. The data are all normalizedto the direct Cy5.5 excitation component and have the Cy3.5, Cy5 andCy5.5 direct excitation components removed to emphasize the resultingsensitization. Only the 1.0×R₀ Holliday/star and 0.5×R₀ constructsmanifest any significant Cy5.5 sensitization. It is also clear fromthese plots that the DNA accurately controls the spacing of these 4 dyesand that the net effect of spacing propagates easily through the 3 stepFRET transfer.

Interestingly, subtracting the directly excited Cy3.5 component in the0.5×R₀ bifurcated structure with all 4 dyes present removes all of itsdetected emission, suggesting that it is executing energy transfer thatis perfect to the limits of measurement. The 0.5×R₀ bifurcated systemalso shows the best AE with a value approaching 300% in Table III. TheAE and E metrics for this data (Table III) also reflect the betterperformance of the 0.5×R₀ construct, while the TEF (FIG. 4A) shows asimilar overall pattern reminiscent of the single FRET step plots inFIG. 2C. Most interesting is that the efficiency is roughly the sameirrespective of the number of arms, indicating that arms act asindependent photonic wires without appreciable exciton transfers betweenthem. Finally, these data show that the sensitization of the terminalacceptor can still be enhanced by at least 30-40× despite threeintervening FRET steps if the number of donor wires and their geometricarrangement are optimized.

In a further generalization of the star antennae geometry, addadditional levels of branching were added as dendrimeric systems. Thesebegin to have densities of chromophores that are reminiscent of naturallight-harvesting complexes. [[Cy3_(n)→Cy3.5]_(n)→Cy5]_(n)→Cy5.5dendrimeric DNA scaffolds were designed such that each dye preceding theCy5.5 was sensitized by either of 2:1, 3:1, or 4:1 donors:acceptor, andwith dye spacings maintained at 0.5×R₀ for high efficiency. This shortinter-dye requirement resulted in structures that grew in complexity anddensity with initial ratios of Cy3:Cy5.5 donor ratios growingexponentially from 8 to 27 to then 64 as shown in FIG. 1C. FIG. 3Fcompares normalized spectra (with the direct excitation componentssubtracted) as collected from the final dendrimer structures assembledwith all dyes present. The spectral profiles tend to be bimodal withinitial Cy3 emission increasing with valence and a minimal contributionof Cy3.5 emission in the 2:1 and 4:1 structures. The sensitized Cy5.5component also appears to be most prominent in the 3:1 assembly with anAE value approaching 400% and the highest E value of the three at 23%(Table III). Here, we estimate formation efficiency at 58%, 84% and 66%for the 2:1, 3:1, and 4:1 structures, respectively, based on FPLCanalysis (SI) and attribute any deficiencies in formation to thecomplexity of the designs.

The 2:1 dendrimer design was extended by adding an initial AF488 dyedonor to create an [[AF488_(n)→Cy3]_(n)→Cy3.5]_(n)→Cy5/AF647]_(n)→Cy5.5construct and assembled two variants with either Cy5 or AF647 present inthe penultimate step. FIGS. 3G and 3H show spectra collected from thefinal dendrimer structures assembled with all dyes present. Despite theadded FRET step, E values are comparable to the previous dendrimers at19% (Cy5 construct) and 16% (AF647) while AE yields are 180% and 140%,respectively.

FIGS. 4A-4D show energy transfer in the photonic wire and dendrimersystems. FIG. 4A is a plot of the Cy5.5 terminal enhancement factor(TEF) for the [Cy3→Cy3.5→Cy5]_(n)→Cy5.5 photonic wire and the 2:1, 3:1,and 4:1 0.5×R₀ dendrimer structures as compared to the initial 1.5×R₀linear system. Note the break in vertical scale. FIG. 4B shows acomparison of the normalized emission profiles for the 0.5×R₀ 2:1dendrimer and 8-arm photonic wire star structures. Dye ratioscorresponding to each position in each structure are indicated with redor blue. Note the significant deconvolved Cy5.5 sensitization for the2:1 dendrimer-inset. FIG. 4C is a comparative plot of the sensitizedcomponents at each step for the [Cy3→Cy3.5→Cy5]_(n)→Cy5.5 photonic wiresystem. Dye emissions are scaled and normalized to the highestcomponent, the Cy3.5 sensitized emission in the 8-arm star structure.FIG. 4D is a comparative plot of the sensitized components at each stepfor the 0.5×R₀ 2:1, 3:1, and 4:1 dendrimer system. Dye emissions arescaled and normalized to the highest component, the Cy3.5 sensitizedemission in the 4:1 structure.

FIGS. 5A-5D show comparisons of “ideal” simulations with experimentalspectra for full 4-dye photonic wire structures with 1, 2, 4, and 8 armsat 0.5×, 1.0×, and 1.5×R₀, and for dendrimers with branching ratios of2:1, 3:1, and 4:1 (0.5×R₀). These simulations show that multipleparallel interacting pathways are better than independent pathways. Whenphotonic wire dye spacing is 1.5×R₀ (FIG. 5A), “ideal” simulations areseen to be in excellent agreement with data, which is not surprisinggiven the weakness of the FRET. At 1.0×R₀ dye spacing (FIG. 5B)agreement is again good for Cy3 and Cy3.5 emission, but less so for theother dyes and especially Cy5.5. Finally, for dye spacings of 0.5×R₀,“ideal” simulations of both the photonic wires (FIG. 5C) and dendrimers(FIG. 5D) do not show a good march to the observed spectra.

For the “low-yield” modeling, the simulated ensembles were taken to bemade up of target structure plus one or more partial structures, withall unincorporated dyes treated as “free.” For simplicity, partialstructures were restricted to having each dye in full complement butwith fewer dye types present, approximating the composite contributionof a wide variety of potential non-fully-formed structures. With thisapproach, one obtains excellent agreement with experiment, and tointerpret the results target structure yields derived in this way (with¾ dyes) were compared to those estimated from gel electrophoresis inFIG. 6A. In general, yield characteristics for the photonic wire anddendrimer structures are similar and suggest a common failure mechanism.That the yields for the two-dye structures (not shown) and for the total(target+partial) product for all structures are uniformly high indicatesthat the Cy3 and Cy3.5 dyes assemble with high fidelity and that theobserved “non-ideal” behavior is due entirely to the Cy5 and/or Cy5.5dyes. Moreover, since simulated yields with 3- and 4-dyes are similar,Cy5 becomes the likely culprit since it must function properly fordownstream Cy5.5 to do so. The decline of assembly yield with increasedstructural complexity, suggests a “crowding” effect due to eitherimpaired hybridization and/or poor Cy5 properties/(self)quenching asnoted before. That performance did not improve when Cy5 was replacedwith AF647 in the 5-dye 2:1 dendrimer (FIG. 1D, FIG. 3G,H) may indicatean assembly problem.

Presuming “low-yield” simulations constitute a plausible understandingof system photophysics, their actual and ideal efficiencies wereestimated along with gain parameters. In FIG. 6B, E is plotted forfour-dye wires as a function of arm number, with ideal results showingthe expected strong boost in efficiency as dye spacing is reduced. Thatthe ideal curves are relatively flat indicates that the arms act mostlyindependently supporting our previous conjecture. Actual efficienciesare greatly reduced in the 1.0× and 0.5×R₀ cases by the yield issuesalready discussed. End-to-end dendrimer efficiencies are shown in FIG.6C with the low “actual” values again resulting from poor yield. In theideal case, efficiency rises with increasing branching ratio by about30%, although the 3:1/4:1 cases are not especially different. The reasonfor both the rise and saturation are the parallel paths in thestructure. To investigate further, within FIG. 6C we show results fromadditional simulations in which FRET was variously restricted. When onlynearest-neighbor dye couplings were included (inset, left side), noefficiency enhancements due to branching were observed. When couplingswere instead restricted to only among dyes on the same branch (inset,right side), then a large fraction of the full ideal curve was realized.Thus, both intra- and inter-branch parallel paths contribute toefficiency enhancement making the dendrimers inherently more efficientthan the photonic wire constructs where the arms act largelyindependently. The antenna properties using an antenna gain (AG) metricanalogous to TEF but relative to the equivalent linear photonic wire(i.e., equal dye spacing) were also examined. Both “ideal” and “actual”AG for the four-dye photonic wire and dendrimer structures are plottedin FIG. 6D. The ideal curve for the wires is close to the unity slopeexpected if all arms operated independently; the slightly higher slopereflects a small contribution from parallel paths. Actual AG is muchlower, again because of yield issues. For the dendrimers, there ispotential for dramatic (exponential) increases in collection with the4:1 structure ideally producing a gain of nearly 400. Yield again causesAG realized to be worse, with the 4:1 dendrimer AG exhibiting a decline.

The multiple interacting parallel FRET pathways as found in thedendrimer deliver more energy and thus more signal for any potentialsensing versus multiple independent pathways as found in the linear andHolliday-star systems.

The following section on a sensor embodiment also includes exemplarysequences used to create a dendrimer scaffold.

Nanoscale Antenna as a Sensor

Including a “toehold” region in the nucleic scaffold allows for sensorsoperable on the basis of complementarity. FIGS. 7A-7D schematicallyillustrate an exemplary dendrimer-based DNA sensor. As can be seen, aportion of the scaffold incorporating an intermediate fluorophore (inthis case, those elements incorporating Cy3.5) includes a toeholdsequence that is not 100% hybridized with the other elements of theassembled scaffold via nucleic acid complementarity. Thus, upon contactwith a sequence a high degree of complementary to the toehold sequence,this portion departs the scaffold (it can be washed away or may simplydepart by diffusion), resulting in a measurable change in photophysicalproperties.

Although FIGS. 7A-7D illustrate only a single toehold sequence in thescaffold, namely on the elements containing the Cy3 dye, a singlescaffold can have more than one distinct toehold sequence. For example,the exemplary sequences below have multiple toehold positions whichcould allow for a more pronounced change in response upon binding of acomplement sequence. In embodiments with more than one scaffold elementhaving a toehold sequence, the toehold sequences may be the same ordifferent within a single scaffold

The following sequences were used to create the nanoscale antennadepicted in FIGS. 7A-7D. In each case, the sequences AGGGAACGAA (SEQ IDNo: 11) and AAGTGCATC (SEQ ID No: 12) are the toehold regions:

21den5s 36 (SEQ ID No: 13):AGGGAACGAA/Cy3/AGAAGAGACAGGGAGT/Cy3/AAGTGCATC21den5s 58 (SEQ ID No: 14):AGGGAACGAACTCCCTGTT/Cy3.5/ACGACCCAGAAGTCACGGGAT/Cy3.5/TCTCTTCTAATGTGCATC 21den5s 76A 2Cy5 (SEQ ID No: 15):AGGGAACGAACTCCCTGTATCCCGTGAC/Cy5/TAACTCGTGAGTGCGGCA/Cy5/CTGGGTCGTATCTCTTCTAATGTGCATC 21den5s 76B Cy55 (SEQ ID No: 16):AGGGAACGAACTCCCTGTATCCCGTGACTTGCCGCAC/Cy5.5/CACGAGTTATCTGGGTCGTATCTCTTCTAATGTGCATC21den5s 58 complementary sequence (SEQ ID No: 17):GATGCACATTAGAAGAGAATCCCGTGACTTCTGGGTCGTAACAGGGAGTT CGTTCCCT21den5s 76A complementary sequence (SEQ ID No: 18):GATGCACATTAGAAGAGATACGACCCAGATGCCGCACTCACGAGTTAAGTCACGGGATACAGGGAGTTCGTTCCCT

As seen in FIGS. 8A and 8B, the resulting amplification-like effect ofthe nanoscale antenna results in an enhanced signal with potentialutility in sensor applications. Namely, by contacting such an antennawith an analyte and exciting the antenna, the degree of response of theantenna to the excitation can indicate a degree of presence in theanalyte by the sequence complementary to the toehold sequence.

A sensor could include, for example, an array of such antennae, andoptionally apparatus to excite and/or measure the response thereof.

Advantages

Techniques described herein use nucleic acid to pattern moleculescapable of energy transfer at specified distances such that the energytransfer is controlled, enabling multi-step energy transfer with controlover the discrete number of FRET steps. This allows for easyreconfiguration of the energy transfer network through the changing of:(a) number of molecules involved in each energy cascade; (b) the numberof linear cascading branches that funnel to a single energy acceptor;and/or (c) the number of donor/acceptor molecules involved in eachdiscrete energy transfer step. The modular design allows for multipleconfigurations within the same building set.

Nanoscale antennas as described herein can be used with other moleculardonor/acceptor pairs beyond the fluorescent dyes used in the examples,for example quantum dots and/or other optically active materials. Thescaffold can be built in two or three dimensions and have theflexibility to incorporate a wide range of dues or other fluorophoresany in a highly controllable configuration.

The technique can involve multiple individual FRET pathways, multipleparallel pathways, or both, and can direct light energy inwards,outwards, or both, or alternatively, in a desired direction. Oneembodiment may have one or more terminal acceptors act as donors in FRETfor a post-terminal acceptor, away from the central position.

A nanoscale antenna can incorporate multiple identical or differentfluorophores at each step to augment energy flow.

Nanoscale antennas have the potential to allow longer range FRETinteractions to compensate for deficiencies in structure or fluorophorefunction. They can harvest light energy one of a wide variety ofwavelengths of choice, or at multiple wavelengths and have potentialutility in optical coding, information storage, information processing,data encryption and sensitization for energy conversion. The techniquesdescribed herein also have utility as a research tool for studyinglight-harvesting (for example in synthetic mimics of photosynthesis).

Although the examples used DNA, the scaffold can incorporate many othernucleic acids and derivatives including RNA, PNA, LNA, etc. Thenanoscale antennas can be easily integrated with other optically activematerials such as, metal chelates, polymers, quantum dots or goldclusters, and furthermore can be designed to be responsive to change intemperature or ionic strength by changing its configuration orhybridization properties, this may also alter FRET properties.

The nanoscale antennas can be arranged on or incorporate active DNAstructures that undergo functional rearrangements in response toexternal stimuli (e.g. another DNA or enzyme) which leads to arearrangement or alteration in ET processes. Moreover, they can be usedto create complex sensing devices such as those capable of detectingchanges in pH (by configuring aspects of the antenna to respond to pH).They can incorporate photoactivatable groups that allow light stimulusto drive subsequent events such as a cleavage for drug delivery.

The nanoscale antennas are expected to have a high degree ofbiocompatibility and can be utilized for biological imaging with reducedbackground from direct excitation. They could function in a time-gatedmodality assuming insertion of appropriate fluorophores with requisiteexcited state lifetimes, and/or in direct excitation or multiphotonmodalities.

The technique described herein allows for antennas that can bebiochemically modified to allow site-specific conjugation to otherbiological or abiotic moieties of interest (e.g. a fluorescent proteinor drug). Moreover, they can be site-specifically modified to allow (a)tethering of the entire structure to an electrode or desired surface (asin a sensor application), optionally through the use of specific bindingpeptides (e.g. polyhistidine, or polycysteine).

A further advantage is that, as implemented here, the technique utilizesa “one-pot” assembly strategy without requiring subsequent purificationsteps. It can be utilized in both ensemble mode or for single-moleculeapplications. Additionally, the nanoscale antennas can be assembled withor incorporate other types of DNA architectures besides the discretestructures utilized here such as DNA origami.

CONCLUDING REMARKS

All documents mentioned herein are hereby incorporated by reference forthe purpose of disclosing and describing the particular materials andmethodologies for which the document was cited.

Although the present invention has been described in connection withpreferred embodiments thereof, it will be appreciated by those skilledin the art that additions, deletions, modifications, and substitutionsnot specifically described may be made without departing from the spiritand scope of the invention. Terminology used herein should not beconstrued as being “means-plus-function” language unless the term“means” is expressly used in association therewith.

REFERENCES

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Ohya, Y.; Yabuki, K.; Hashimoto, M.; Nakajima, A.; Ouchi, T. Bioconjug.Chem. 2003, 14, 1057

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Hannestad, J. K.; Sandin, P.; Albinsson, B. J. Am. Chem. Soc. 2008, 130,15889

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TABLE I Fluorophore photophysical and FRET properties. λ_(max) Ext.coeff. abs. λ_(max) ¹R₀ in Å/J(λ) in cm³M⁻¹ Fluorophores QY (M⁻¹cm⁻¹)(nm) em. (nm) AF488 Cy3 Cy3.5 Cy5 AF647 Cy5.5 AF488 0.39 71,000 495 51946/1.23e⁻¹³ 61/6.94e⁻¹³ 59/5.93e⁻¹³ 49/1.90e⁻¹³ 47/1.42e⁻¹³ 43/8.92e⁻¹⁴Cy3 0.15 150,000 550 570 — 47/3.68e⁻¹³ 53/8.01e⁻¹³ 54/9.37e⁻¹³53/7.83e⁻¹³ 49/4.73e⁻¹³ Cy3.5 0.15 150,000 581 596 — — 44/2.70e⁻¹³60/1.69e⁻¹² 59/1.58e⁻¹² 55/1.01e⁻¹² Cy5 0.28 250,000 649 670 — — —65/1.39e⁻¹² — 68/1.94e⁻¹² AF647 0.33 239,000 650 665 — — — — 65/1.17e⁻¹²72/2.18e⁻¹² Cy5.5 0.23 190,000 675 694 — — — — — 63/1.41e⁻¹² ¹R₀ andJ(λ) values are averages calculated from the spectra of all dye-labeledDNA used in this study. QY—quantum yield.

TABLE 2 Antenna effect (AE) and end-to-end efficiency (E) for theCy3-Cy5 single FRET step system. Förster distance/donor-acceptorseparation 0.75/40.5 Å 0.85/45.9 Å 1.0/54 Å 1.25/67.9 Å 1.5/81 ÅConstruct (#Cy3/Cy5) ε_(Cy3n)/ε_(Cy5) ¹ AE (%)/E (%) AE/E AE/E AE/E AE/ELinear (1) 0.6 108/40  76/22  54/18 34/9 16/3 Bifurcated (2) 1.2 185/51128/28  84/20  55/10 30/4 Holliday junction (4) 2.4 291/37 120/30 144/2383/9 31/4 8-arm star (8) 4.8 252/15 163/16 183/15 80/8 46/2 All valuesare collected from at least 3 independently assembled structures.Standard deviations for AE and E values are all <10%. ¹Initial Cy3_(n)absorption at 550 nm relative to the final Cy5 absorption at 650 nm.

TABLE 3 Antenna effect (AE) and end-to-end efficiency (E) for the4/5-dye photonic wire and dendrimer systems. Förster distance 0.5 × R₀^(1,2) 1.0 × R₀ 1.5 × R₀ Construct (#wires³/Cy5.5) ε_(Cy3n)/ε_(Cy5.5) ⁴AE (%)/E (%) AE (%)/E (%) AE (%)/E (%) Linear 1 0.8 184/16 46/4 17/2Bifurcated 2 1.6 285/14 85/7 13/1 Holliday junction 4 3.2 107/9  73/921/3 8-arm star 8 6.3 113/6  78/6 34/2 4-dye (#Cy3/Cy5.5) 2:1 dendrimer8 6.3 217/17 — — 3:1 dendrimer 27 21.3 393/23 — — 4:1 dendrimer 64 50.5158/8  — — εAF488n/ 5-dye dendrimer (#AF488/Cy5.5) εCy5.55 2:1 (Cy5)⁶ 166 180/19 — — 2:1(AF647)⁶ 16 6 140/16 — — All values were collected fromat least 3 independently assembled structures. Standard deviations forAE and E values are all <10%. ¹See Table 1 for individual dye-dye R₀values. ²Standard deviations of all values <10%. ³Wire =[Cy3→Cy3.5→Cy5]_(n). ⁴Initial Cy3_(n) absorption at 550 nm relative tothe final Cy5.5 absorption at 700 nm. ⁵Initial AF488_(n) absorption at550 nm relative to the final Cy5.5 absorption at 700 nm. ⁶Displayingeither Cy5 or AF647 at the 4^(th) dye.

What is claimed is:
 1. A nanoscale antenna comprising: a nucleic acidscaffold having a structure selected from the group consisting of aHolliday junction, a star, and a dendrimer; and a plurality offluorophores attached to the scaffold and configured as a FRET cascadecomprising at least three different types of fluorophores, arranged with(a) a plurality of initial donor fluorophores fixed in exteriorpositions on the structure, (b) a terminal acceptor fluorophore fixed ina central position on the structure, and (c) a plurality of intermediatefluorophores fixed in positions on the scaffold between the initialacceptor fluorophores and the terminal acceptor fluorophore, wherein oneor more portions of said scaffold incorporating intermediatefluorophores further comprise a toehold sequence, and are detachablefrom said scaffold upon contact with a sequence complementary to thetoehold sequence, and wherein the plurality of fluorophores comprises atleast one form of quantum dot.
 2. The nanoscale antenna of claim 1,wherein said nucleic acid is DNA and wherein at least one of thefluorophores is a fluorescent dye integrated into the DNA viaphosphoramidite chemistry; succinimidyl ester chemistry; maleimide thiolchemistry; a carboxyl-amine amide bond; azide-alkyne cycloaddition; or acombination thereof.
 3. The nanoscale antenna of claim 1, wherein saidFRET cascade includes a total of 3, 4, 5, or 6 different types offluorophores.
 4. The nanoscale antenna of claim 1, wherein said terminalacceptor comprises one or more fluorophores configured donors in FRETfor a post-terminal flurophore acceptor located away from said centralposition.
 5. A nanoscale antenna comprising: a nucleic acid scaffoldhaving a dendrimer structure, and a plurality of fluorophores attachedto the scaffold and configured as a FRET cascade comprising at leastthree different types of fluorophores, arranged with (a) a plurality ofinitial donor fluorophores fixed in exterior positions on the structure,(b) a terminal acceptor fluorophore fixed in a central position on thestructure, and (c) a plurality of intermediate fluorophores fixed inpositions on the scaffold between the initial acceptor fluorophores andthe terminal acceptor fluorophore, wherein one or more portions of saidscaffold incorporating intermediate fluorophores further comprise atoehold sequence, and are detachable from said scaffold upon contactwith a sequence complementary to the toehold sequence, and wherein theplurality of fluorophores comprises at least one form of quantum dot. 6.The nanoscale antenna of claim 5, wherein said nucleic acid is DNA andwherein at least one of the fluorophores is a fluorescent dye integratedinto the DNA via phosphoramidite chemistry; succinimidyl esterchemistry; maleimide thiol chemistry; a carboxyl-amine amide bond;azide-alkyne cycloaddition; or a combination thereof.
 7. The nanoscaleantenna of claim 5, wherein said FRET cascade includes a total of 3, 4,5, or 6 different types of fluorophores.
 8. The nanoscale antenna ofclaim 5, wherein said terminal acceptor comprises one or morefluorophores configured donors in FRET for a post-terminal flurophoreacceptor located away from said central position.
 9. A method of using ananoscale antenna, the method comprising: providing a nanoscale antennacomprising: a nucleic acid scaffold having a structure selected from thegroup consisting of a Holliday junction, a star, and a dendrimer; and aplurality of fluorophores attached to the scaffold and configured as aFRET cascade comprising at least three different types of fluorophores,arranged with (a) a plurality of initial donor fluorophores fixed inexterior positions on the structure, (b) a terminal acceptor fluorophorefixed in a central position on the structure, and (c) a plurality ofintermediate fluorophores fixed in positions on the scaffold between theinitial acceptor fluorophores and the terminal acceptor fluorophore,wherein the plurality of fluorophores comprises at least one form ofquantum dot and wherein one or more portions of the scaffoldincorporating intermediate fluorophores further comprise a toeholdsequence detachable from said scaffold upon contact with a sequencecomplementary to the toehold sequence; contacting the nanoscale antennawith an analyte; exciting the antenna with a light source to excite theFRET cascade; and measuring a response of said nanoscale antennafollowing the excitation, wherein the response indicates a degree ofpresence in the analyte of the sequence complementary to the toeholdsequence.
 10. The method of claim 9, wherein said nucleic acid is DNAand wherein at least one of the fluorophores is a fluorescent dyeintegrated into the DNA via phosphoramidite chemistry; succinimidylester chemistry; maleimide thiol chemistry; a carboxyl-amine amide bond;azide-alkyne cycloaddition; or a combination thereof.
 11. The method ofclaim 9, wherein said FRET cascade includes a total of 3, 4, 5, or 6different types of fluorophores.